Method of increasing microbial activites



METHOD DE EN CREASDJG MICROBIAL ACTHVITIES Elmer A. Weaver, Spring Mount, Pa., assignor to the United States of America as represented by the Secretary of Agriculture No Drawing. Filed Mar. 8, 1960, Ser. No. 13,670

6 Claims. (Cl. 195-51) (Granted under Title 35, US. Code (1952), see. 266) A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to a procedure for microbial transformation of high concentrations of comparatively insoluble substrates, in particular relating to the microbiological oxidation of progesterone.

Previous attempts to employ high concentrations of steriod substrates, such as progesterone, in contact with microorganisms have encountered various difiiculties. When the steroid is added as a solution in a suitable solvent such as propylene glycol, a limiting factor on the use of high concentrations is the toxicity of the solvent. When the steroid is added as a suspension in water, a serious problem is the physical damage to the microbial cells by the high concentration of steroid particles, even when the steroid is finely ground or micronized by conventional grinding or crushing procedures.

Thus, physical properties of a solid substrate are a critical limitation in employing high concentrations of substrate in a fermentation. Solid masses of the particles and individual particles should not be capable of damaging action to the microbial cells during fermentation through collisions of the cell and substrate. There should be no sharp edges, corners, needle points, or other piercing or cutting protuberances capable of damaging action.

An object of the present invention is to conduct microbiological oxidation of progesterone in the presence of high concentrations of progesterone and ll-alpha-hydroxyprogesterone. Another object is to provide particles of progesterone which are relatively harmless to the micro-organisms in the culture medium. These and other objects are achieved in accordance with the invention described below.

The inventive process is demonstrated by its application to the hydroxylation of progesterone. Raw progesterone as available on the market was used as a control.

Raw progesterone was ground in a mortar and pestle to a very fine particle size equivalent in dimensions, but not in configurations, to the size obtainable for the progesterone smooths as produced below.

Progesterone smooths were prepared by exposing the raw progesterone particles to a violent turbulence created by supersonic velocity air streams, as that provided by various fluid-energy type milling equipment. These high speed airstreams caused attritive collisions of the particles. This attrition action produced particles referred to as smooths. Progesterone smooths meet the physical requirements discussed earlier for substrate preparation,

The progesterone smooths used as substrate in carrying out the process of the inventionn were found to have a total volume several fold over the total volume occupied by the unprocessed raw progesterone. The increase in volume of the progesterone smooths is believed due in most part to the many longitudinal splits of the progesterone crystals resulting in flake formation. Many flakes derived from one crystal are thus able to fluff up the volume due to random distribution. These flakes or sheets ted grates Patent become evident in the progesterone smooths by their ability to transmit amounts of light greater than crystals of equal area found in raw progesterone.

Microscopical examination of both raw and smooths progesterone samples disclosed the presence of fines (less than ten microns) in both samples ranging up to 20 or 30% of the total mass present. However, the fines in the raw progesterone were found to have irregular and rough surfaces with many needle sharp points. The fines from the progesterone smooths were found to have regular smooth surfaces and an absence of needle sharp points.

The same general situation was found to exist for the larger particles in both the raw progesterone, mostly 30 to 150 microns on longest axis, and the progesterone smooths, mostly 30 to 50 microns on longest axis.

The mortar and pestle ground raw progesterone, although having particles reduced in size similar to progesterone smooths, were found to have the surface features of the initial raw progesterone.

The progesterone smooths are better able surface contact with the smooth surface of the microbial cell. This high degree of interfacial contact promotes faster reaction rates between substrate and microbial cell complexes involved in transformation reactions. This is an especially valuable feature in reactions wherein contact of substrate and microbial cell is required.

According to the present invention the hydroxylation of high concentrations of progesterone, in the range of 20 to 50 grams per liter and above, is obtained by a process comprising the preparation of progesterone smooths, wetting down the selected amount of progesterone with water containing 0.01% of a Wetting agent (such as Tween 80, chemically identified as polyoxyethylene sor bitan mono-oleate) in about a 1:1 volume in a cotton stoppered flask, heating the flask and contents in live steam at atmospheric pressure for 30 minutes, cooling, aseptically adding an aliquot of a vigorously growing culture, such as Aspergillus ochraceus, diluting the culture with at least about an equal volume of sterile, distilled water, incubating the diluted culture for about 24 to 72 hours and recovering the hydroxylated progesterone (ll-alphahydroxyprogesterone).

It is important that the microbial culture providing the oxidizing enzymes be grown under conditions resulting in rapid cell development. This can be done by multiple successive transfers of the organism in stock culture while employing a favorable growth medium. The medium for vegetative growth of the organism contained 2% Edamin (lactalbumin enzymatic digest), 5% dextrose and 0.63%

corn steep liquor, and was adjusted to pH 5.5.

The minimum dilution at which the process will Work efiiciently is considered to be the addition of about an equal volume of distilled water. The dilution step is conveniently performed at the time the culture is added to the substrate. Preferable operating range for dilution is considered to be between one and twenty-five dilution volumes.

The particular wetting agent used to facilitate suspension of the high concentrations of the especially prepared progesterone smooths" is not critical to the process as long as the agent is not toxic to the microorganism. Materials other than Tween type compounds which may be used are any known surface depressant agents in low concentration such as water solutions of ethanol, methanol, and the like.

The present invention is illustrated by the following examples.

it a

to make EXAMPLE 1 This example compares results with progesterone as available commercially (referred to as raw), raw

progesterone ground in a mortar and pestle to a fine particle size, and progesterone smooths prepared as previously described. Solvent addition of progesterone could not be tried due to the high level of progesterone employed. Quantities of solvent carriers needed to dissolve this high concentration of progesterone would be inhibitory to activities of the microbial cells.

The reaction flasks were prepared by adding 2.5 grams progesterone to each 250 ml. Erlenmeyer flask. The pro gesterone smooths after weighing into the flask were wetted down with a small amount of 0.01% Tween 80 water solution and the flasks plugged with cotton batting. This treatment made it possible to form a uniform suspension and eliminated floating balls of progesterone smooths which had entrapped air. The other samples of progesterone did not require wetting down with the Tween 80 solution but were similarly treated for control purposes. The flasks were then exposed to live steam at atmospheric pressure for 30 minutes, After cooling, 25 mls. of an abundant growth of Aspergillus ochraceus NRRL 405 were aseptically added to the flask plus 25 mls. of sterile, distilled water. The flasks were incubated at 28 degrees cent. for 72 hours on a rotary shaker set at 220 rpm. and a stroke of two inches.

At the end of the incubation time, all samples were extracted 7 times with equal volumes of chloroform. The chloroform was evaporated to dryness and the residue picked up in hot benzene. Skelly C was added to the hot benzene to incipient precipitation and then cooled for continued crystal formation. All crops of crystals melt ing 150-160 degrees C. were combined and recrystallized from Skelly C-methanol. All crops melting 159 to 161 degrees C. and having a Rf value the same as pure 11ahydroxyprogesterone were combined as total yield of 1 la-hydroxyprogesterone.

Raw progesterone gave a yield of 41% lle-hydroxyprogesterone while the fine particles from the motar and pestle ground progesterone gave a yield of essentially the same amount at 43.5%. The progesterone smooths in this same test gave a yield of 64% lla-hydroxyprogesterone.

It is thus seen that a combination of dilution growth of microbial cells plus a high concentration of specially prepared progesterone particles (smooths) are able to accomplish high conversion of progesterone to the desired end-product of lla-hydroxyprogesterone.

Simple reduction of the solid progesterone particles to a fine particle size by ordinary grinding or comminution is not suflicient to accomplish the purposes of the inventive process as is shown by the results of this example.

EXAMPLE 2 This example demonstrates the need for the dilution step in the process. 2% progesterone smooths were used as the substrate. The same organism and incubation conditions as given in Example 1.

25 mls. of growth plus 25 mls. of water plus one gram of progesterone smooths in a 250 mls. Erlenmeyer flask incubated for 72 hours yielded 87% lla-hydroxyprogesterone. In another experiment, 250 mls. of growth plus 250 mls. of distilled water plus grams of progesterone smooths in a 4 liter serum bottle incubated for 72 hours yielded 90% lle-hydroxyprogesterone. A similar experiment with no dilution of the medium, i.e. 50 mls. of growth, plus one gram of progesterone smooths in each 250 Erlenmeyer flask incubated for 72 hours yielded only 74% lla-hydroxyprogesterone. It is therefore evident that the diluted cultures. give similar results and further confirm the high yields obtainable with the combination of dilution growth plus specially prepared progesterone smooths.

EXAMPLE 3 This example shows the inhibitory action of the sub strate (progesterone) when it is contacted with the or- Papergram Weight inresults, percrease, rr Progesterone Treatment cent 11-11 dry microbial hydroxypro cells gesterone A. Control-none B. Raw prog C. Mortar and pestle ground pro D. Prog. smooths The inhibitory action on the growth of the mycelium, demonstrated to be present whether the progesterone solid particles are comparatively large as in raw progesterone (B) or whether they are comminuted to a fine particle size such as can be done by grinding the progesterone .with a mortar and pestle (C), is attributed to physical damage to the microorganism caused mainly by the surface configurations of the progesterone particles. Inhibitory action due to the physical state of the solid progesterone particles has been practically eliminated by the treatment as illustrated by results obtained for the progesterone smooths (D).

EXAMPLE 4 Rate of hydroxylation This example showes relationship of the present process to the best reported process for high speed production of desired end-product. The high levels of substrate concentration possible with the treatment according to this invention does not permit a direct comparison with addition of the substrate in a solvent carrier. However, the process can be compared with results from using progesterone smooths and semi-continuous solvent carrier addition for the substrate as illustrated by the report given in Ind. Eng. Chem. 48, 2213 (1956). This latter report (semicontinuous IEC) gives details on the highest concentration of progesterone used in a fermentation heretofore. Aspergillus ochraceus was used in this referenced report as well as for the results reported herein. The organism was grown and used as given in Example 1. In the IEC report the organism was grown similarly but did not receive the dilution step. The Rate of Hydroxylation table below gives other details and results of the experiments.

RATE OF HYDROXYLATION Mgs. of 11a Percent Incubahydroxypro- Progesterone Prog. tion gesterone Time Per mls.

Per Hour Smooths 5.0 72 44 Semi-eontinuous IEC r 0. 4 28 11 "Smooths 2.0 24 58 The rate of the transformation reaction promoted by the present process is more than five times faster than the tion time of 72 hours, the new process is four times as fast as the semi-continuous solvent addition process and the concentration of the substrate more than twelve times as great.

The ease with which the product can be recovered is illustrated in the following example in which a 2% suspension of progesterone smooths was used as the substrate and employing the same organism and incubation conditions as given in Example 2.

EXAMPLE 5 A volume of 25 mls. of abundant growth of the organism plus 25 mls. of water plus one gram of progesterone smooths were placed in each of two 250 ml. Erlenmeyer flasks and incubated 72 hours. At the end of the incubation period, 2% by weight Supercel filter aid was added to one flask while nothing was added to the other. The entire contents of each flask was easily suction filtered through No. 1 Whatman filter paper. Both filtrates were free of mycelium. The Supercel was used to see if it would contribute to more product retention in the filter cake or make the filtration easier.

The filter cake of each sample was re-suspended in 50 mls. of acetone and steeped 24 hours. The mixture was then filtered to give the first acetone extract. It was repeated to give the second acetone extract. The filter cake was finally given a hot chloroform extract in a similar manner.

The filtrate was extracted three times with one half volumes of chloroform followed by three extractions using equal volumes of ethyl acetate. All extracts were combined to give the filtrate extract.

Extract weights in milligrams were as follows:

The results show that about 95% of the recoverable product is in the filter cake. Further, the results show that most of the product can be recovered by a single extraction of the filter cake. Supercel is not necessary in the recovery of the product.

The first acetone extracts of both untreated and Supercel treated were combined for crystallization work. Two re-crystallizations from hot benzene and Skelly C yielded a weight of 1.7284 grams with MP. 158-166. Papergram results showed the product to be identical with ll-alphahydroxyprogesterone.

The filter cake occupies a small volume of the ferment, frequently less than 5% of the total volume depending on the degree of dilution given in the dilution step. Any reacted product recoverable in the filter cake would be more readily available for this reason. Other objectionable features associated with recovery of the product from fiuids such as the filtrate would also be eliminated. Principal difficulties of this kind are emulsion formation, the handling of large volumes of organic solvents, and much careful and tedious manual work.

Recapitulating, it will be seen that present process has an advantage in that all of the substrate may be added at one time and the fermenter need not be opened again during the fermentation. A concomitant advantage resides in the ease with which the product is recovered by simple filtration as is illustrated in Example 5.

The dilution step required in the process reduces the total weight of microbial cells involved in the reaction to a fraction of that traditionally employed. This results in ii less waste cells for disposal and. less contaminating substances to be removed from the reaction products. The high concentration of the substrate undergoing reaction further minimizes fermentation impurities in the reaction products since the fermentation impurities are independent of substrate concentration.

A further advantage is the unusually high rates of transformation reactions obtainable. This is a unique feature and an unexpected result in the operation of the inventive process. Comparative results with conventional processes is given in Example 4.

Progesterone substrate added to fermentation media in organic solvents immediately forms microscopic sized crystals to give the medium a milky appearance. In this condition the progesterone is uniformly dispersed and presents a large surface area for substrate action. This is highly desirable. However, these crystals grow in a very short time (frequently minutes) into large crystals which sink to the bottom leaving a clear solution above. These large crystals present a comparatively small surface area for substrate action. Of particular advantage is the amorphous condition of the progesterone smooths which cause them to retain their original physical state until the microbiological transformation reaction is completed.

In the practice of this invention it was discovered that generation of products other than the desired product was reduced below significant levels or eliminated entirely from the reaction. Observation of the examples discloses mono-hydroxylation in high yield, yet, polyhydroxylation of progesterone was not evident even though an organism, Aspergillus ochraceus, known for its ability to produce poly-hydroxylation, after the introduction of one hydroxyl group, was used. By-product formation is thus prevented as well as wasteful loss of the substrate in these side reactions.

I claim:

1. An improved process for the microbiological oxidation of progesterone to l1-alpha-hydroxyprogesterone which comprises suspending smooth-surfaced particles produced by attritively colliding crystalline particles of progesterone at supersonic velocity in water containing a small amount of a wetting agent, sterilizing the suspension, adding a quantity of the sterilized suspension to a vigorously growing culture of Aspergillus ochmceus diluted with at least about an equal volume of water to provide a concentration of progesterone of about from 20 to 50 grams per liter of diluted culture, incubating the resulting mixture whereby progesterone is oxidized by the growing culture to ll-alpha-hydroxyprogesterone, and recovering the 1l-alpha-hydroxyprogesterone from the reaction mixture.

2. An improved process for the microbiological oxidation of progesterone to l1-alpha-hydroxyprogesterone which comprises attritively colliding crystalline particles of progesterone at supersonic velocity to produce relatively smooth-surfaced particles, suspending the smoothsurfaced particles in water containing a small amount of a wetting agent, sterilizing the suspension, adding to the sterilized suspension a growing culture of Aspergillus chraceus, then incubating said suspension whereby progesterone is oxidized by the growing culture to ll-alphahydroxyprogesterone, and recovering ll-alpha-hydroxyprogesterone from the reaction mixture.

3. An improved process for the microbiological oxidation of progesterone to ll-alpha-hydroxyprogesterone which comprises attritively colliding crystalline particles of progesterone at supersonic velocity to produce relatively smooth-surfaced particles, suspending the smoothfaced particles of progesterone in water containing a small amount of a wetting agent, adding to said suspension a growing culture or" Aspergfllus oclzraceus, and then incubating said suspension whereby progesterone is oxidized by the growing culture to 1l-alpha-hydroxyprogesterone.

4. The process of claim 3 wherein the smooth-surfaced particles are produced from crystalline particles of progesterone by attritively colliding said crystalline particles while suspended in a turbulent air stream. moving at supersonic velocity.

5. An improved process for the microbiological oxidation of progesterone to 1l-alpl1a-hydroxyprogesterone which comprises attritively colliding crystalline particles of progesterone at supersonic velocity to produce relatively smooth-surfaced particles, suspending the smoothsurfaced particles in water containing a small amount of a wetting agent, sterilizing the suspension, adding a quantity of the sterilized suspension toa vigorously growing culture of Aspergillus ochraceus diluted with at least about an equal volume of water to provide a concentration of progesterone of about 20 to 50 grams per liter of diluted culture, incubating the resulting mixture whereby progesterone is oxidized by the growing culture to insoluble 11-alpha-hydroxyprogesterone, physically separating insoluble 11-alpha-hydroxyprogesterone together with the microbial cells from the culture medium, extracting the 8 mixture of cells and ll-alpha-hydroxyprogesterone with a solvent for said l1-alpha-hydroxyprogesterone, separating the solution from the cells, and evaporating the solvent to recover 1l.-alpha-hydroxyprogesterone.

6. T he process of claim 5 wherein the solvent is selected from the group consisting of acetone and chloroform.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Perry: Chemical Engineers Handbook, 3rd edition,

1950, the Maple Press Co, York, Pa, pp. 11431l45 TP 

1. AN IMPROVED PROCESS FOR THE MICROBIOLOGICAL OXIDATION OF PROGESTERONE TO 11-ALPHA-HYDROXYPROGESTERONE WHICH COMPRISES SUSPENDING SMOOTH-SURFACED PARTICLES PRODUCED BY ATTRITIVELY COLLIDING CRYSTALLINE PARTICLES OF PROGESTERONE AT SUPERSONIC VELOCITY IN WATER CONTAINING A SMALL AMOUNT OF A WETTING AGENT, STERILIZING THE SUSPENSION, ADDING A QUANTITY OF THE STERILLIZED SUSPENSION TO A VIGOROUSLY GROWING CULTURE OF ASPERGILLUS OCHRACEUS DILUTED WITH AT LEAST ABOUT AN EQUAL VOLUME OF WATER TO PROVIDE A CONCENTRATION OF PROGESTERONE OF ABOUT FROM 20 TO 50 GRAMS PER LITER OF DILUTED CULTURE, INCUBATING THE RESULTING MIXTURE WHEREBY PROGESTERONE IS OXIDIZED BY THE GROWING CULTURE TO 11-ALPHA-HYDROXPROGESTERONE, AND RECOVERING THE 11-ALPHA-HYDROXYPROGESTERONE FROM THE REACTION MIXTURE. 